Therapeutic use of d-methionine to reduce the toxicity of platinum-containing anti-tumor compounds

ABSTRACT

Methods of preventing or reducing hearing or balance loss, damage to ear cells, weight loss, gastrointestinal toxicity, neurotoxicity, alopecia, and prolonging survival in patients undergoing treatment with therapeutically effective amounts of platinum-containing chemotherapeutic agents such as cisplatin are provided. These methods comprise administering an effective amount of a methionine protective agent, such as D-methionine, prior to, simultaneously with, or subsequently to administration of the platinum-containing chemotherapeutic agent. Combinations of these time periods can also be employed.

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 60/027,750, filed Oct. 3, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of protective agents in cancerchemotherapy in human and animal subjects. Protective agents arecompounds that prevent, reduce, or otherwise ameliorate the toxic sideeffects of anti-cancer chemotherapeutic compounds in normal body cellswhile substantially preserving the anti-tumor properties of thesecompounds in vivo when administered prior to, concomitantly with, orsubsequently to administration of such chemotherapeutic compounds. Morespecifically, the present invention relates to the use of D-methionineand structurally related compounds as protective agents havingoto-protective, weight loss-protective, gastrointestinal-protective,neuro-protective, alopecia-protective, and survival-enhancing effects inconjunction with chemotherapy employing platinum-containingantineoplastic agents, such as cisplatin.

2. Description of Related Art

Cisplatin Chemotherapy

Cisplatin (cis-diamminedichloroplatinum(II); CDDP) is a widely usedantineoplastic agent. Cisplatin administration has increased both in thevariety of cancer types for which it is employed and in the amount usedin a given individual to achieve maximal therapeutic effect (Blumenreichet al., 1985; Forastiere et al., 1987; Gandara et al., 1989).

The toxic side effects of cisplatin have long been recognized and arewidely reported (Lippman et al., 1973; also see the review by Hacker,1991). These toxicities include a variety of peripheral neuropathies,myelo-suppression, gastrointestinal toxicity, nephrotoxicity, andototoxicity (Ozols and Young, 1985; Stewart et al., 1987; Stoter et al.,1989). Initially, the primary dose-limiting factor was nephrotoxicity,but now the routine administration of mannitol, hypertonic saline, andhigh fluid administration have ameliorated, but not eliminated, thatside effect. However, ototoxicity remains uncontrolled (Bajorin et al.,1987; Fillastre and Raguenez-Viotte, 1989). Although nephrotoxicity canstill be dose-limiting, currently the primary dose-limiting factor isototoxicity (Blumenreich et al., 1985; Forastiere et al., 1987; Berry etal., 1990).

The primary ototoxic effects of cisplatin appear to occur in thecochlea. Anatomical changes occur in both the stria vascularis and theorgan of Corti. The primary histologic findings include hair celldegeneration and damage to the supporting cells that are dose-related(Anniko and Sobin, 1986). At high doses, total collapse of themembranous labyrinth can occur (Anniko and Sobin, 1986). In the organ ofCorti, there is loss of outer and inner hair cells, with a propensityfor outer hair cell loss in the basal turn (Fleischman et al., 1975;Komune, 1981; Estrem et al., 1981; Schweitzer, 1993), and alterations inthe supporting cells and Reissner's membrane (Komune, 1981; Estrem etal., 1981). Estrem et al. (1981) also reported softening of thecuticular plate and an increased number of lysosomal bodies in theapical portion of the outer hair cell. However, the mechanisms inducingthese changes are largely unknown.

For equivalent inner ear concentrations, cisplatin is the most ototoxicdrug known (Moroso and Blair, 1983; Koegel, 1985; Anniko and Sobin,1986; Griffin, 1988). Generally, cisplatin ototoxicity is irreversible,its onset insidious, and the hearing loss may progress afterdiscontinuation of the protocol (Schaefer et al., 1985; Melamed et al.,1985; Pollera et al., 1988; Aguilar-Markulis et al., 1981; see thereview by Moroso and Blair, 1983). Hearing loss is usually permanent(Vermorken et al., 1983). Partial recovery may occur in some cases, butonly one of 121 patients with hearing loss had complete recovery in astudy by Aguilar-Markulis et al., (1981). Hearing loss typically startsat the ultra high frequencies (9000 to 20000 Hz) (Fausti et al., 1984;Kopelman et al., 1988) and then progresses into the high conventionalaudiometric range (Laurell and Engström, 1989; Kopelman et al., 1988;Meyer, 1989), reducing the patient's ability to hear consonant but notvowel sounds. An inability to understand speech and tinnitus arefrequent complaints (Kopelman et al., 1988). An increasing number ofpatients survive chemotherapy, but frequently with hearing impairment.

Nucleophilic Sulfur Protective Agents

Many sulfur-containing compounds (including substances with thio, thiol,and thioether groups) have been reported to provide CDDPnephroprotection in animal models (Anderson et al., 1990; Jones andBasinger, 1989; Jones et al., 1986; 1991a, b, c; 1992). These compoundsmay act by preventing the CDDP-induced depletion of glutathione or thebinding of CDDP to protein sulfhydryl groups (Hanneman and Baumann,1988; Nakano and Gemba, 1989; Gandara et al., 1989; Ravi et al., 1991;Schweitzer, 1993).

Additionally, sodium thiosulfate (STS) and diethydithiocarbamate (DDTC)provide good CDDP otoprotection in animals (Otto et al., 1988; Church etal., 1995; Rybak et al., 1995). Unfortunately, STS may reduce CDDPtumoricidal action (Pfeifle et al., 1985; Aamdal et al., 1987) and mayexacerbate CDDP-induced weight loss and mortality (Otto et al., 1988).DDTC does not interfere with antitumor action (Qazi et al., 1988; Berryet al., 1989; Dedon et al., 1984; Borch et al., 1988), but can producesevere side effects (Rothenberg et al., 1988; Qazi et al., 1988).

D-Methionine

D-methionine (D-Met) is a sulfur-containing nucleophile that provideshighly effective CDDP nephroprotection in animals without decreasinganti-tumor action (Jones and Basinger, 1989). Although only tested inthat single study at a single dose level, D-Met was the most effectiveCDDP nephroprotectant that did not interfere with CDDP tumoricidalaction out of nearly 40 sulfur-containing agents tested in a series ofstudies by Jones and colleagues (Jones and Basinger, 1989; Jones et al.,1986; 1991a, b, c; 1992). As far as the inventor is aware, D-Met hasnever been previously tested as a CDDP otoprotectant, and has not yetbeen tested clinically (Treskes and van der Vijgh, 1993).

Sulfur-Containing Protective Agents and the Modulation ofCisplatin-Induced Toxicity

Studies indicate that individual sulfur-containing protective agents mayonly be effective in reducing specific types of toxicity, such asnephrotoxicity, while remaining ineffective in blocking otherplatinum-related complications such as peripheral neuropathy andototoxicity (Schweitzer, 1993). In addition, an agent which is effectiveas a regional chemoprotector following site-specific (intraperitoneal)usage of platinum-containing compounds such as CDDP may fail to provideadequate systemic protection, or may inhibit antitumor activity(Schweitzer, 1993).

Not all sulfur-containing compounds provide protection against all ofCDDP's toxicities, and it is not possible to predict which protectiveagents will be effective or ineffective for this purpose. For example,cefoxitin (Jones et al., 1992) does not provide nephroprotection.Ethyl-L-cysteinate and N-(2-mercapto-propionyl)glycine (Jones andBasinger, 1989) exacerbate CDDP nephrotoxicity. 2-(methylthio)nicotinicacid does not provide nephroprotection in rats (Jones et al., 1991b).The sodium salt of penicillin G does not protect against CDDPnephrotoxicity or weight loss (Jones et al., 1992). Similarly,thiamine-HCl does not protect against cisplatin nephrotoxicity or weightloss (Jones et al., 1992).

Furthermore, sulfur-containing compounds protective against one type ofCDDP toxicity frequently do not protect against other CDDP toxicities,and it is not possible to predict the specific antitoxic effectivenessof such compounds. Cephalexin (Jones et al., 1992) protects againstCDDP-induced kidney dysfunction and weight loss, but curiously does notprevent kidney pathology. Cefoxitin (Jones et al., 1992) provides someprotection against CDDP-induced weight loss, but does not protectagainst CDDP nephrotoxicity. The sodium salt of penicillin G does notprotect against either CDDP-induced nephrotoxicity or weight loss (Joneset al., 1992). Sulfathiazole provides protection against CDDPnephro-toxicity, but not weight loss (Jones et al., 1992).

WR2721 provides excellent CDDP nephroprotection (Mollman et al., 1988),but does not ameliorate nausea and vomiting (Glover et al., 1987). Nordoes WR2721 seem to provide CDDP otoprotection: Glover et al. (1987)found mild to severe hearing loss in 20 of 36 patients receiving WR2721prior to CDDP although nephroprotection was obtained. Rubin et al.(1995) reported a 45% incidence of significant hearing threshold shiftin patients pretreated with WR2721 prior to CDDP administration.Unfortunately, neither the Glover et al. (1987) nor Rubin et al. (1995)studies employed a control group, and both reported a high incidence ofototoxicity in patients receiving WR2721. In hamsters, Church et al.(1995) reported no WR2721 protection from ototoxicity or mortality.

Even when a sulfur-containing agent is found to be protective, its sideeffects can be so severe that clinical applicability is precluded. Inaddition, even among agents that provide CDDP otoprotection, theprotection may be so inconsistent and/or the side effects so great thatthey would not be used clinically. For example, DDTC provides protectionagainst CDDP-induced nephrotoxicity (Qazi et al., 1988; Berry et al.,1989; Gandara et al., 1989a, 1989b, and 1991) and ototoxicity (Church etal., 1995), but the protection against ototoxicity may only be partial(Gandara et al., 1989a; Ravi et al., 1992) and its side effects aresevere (Rothenberg et al., 1988; Berry et al., 1990). If DDTC dosing isreduced to ameliorate its side effects, adequate protection from CDDPside effects may not occur (Paredes et al., 1988). Similarly, disulfiram(Antabuse), which can be used as a precursor for its metabolite DDTC,can cause sensorimotor neuropathy (Argov and Mastiglia, 1979) andreversible confusion that can be dose-limiting (Stewart et al., 1987).Consequently, it is unlikely that DDTC will be widely used clinically asa CDDP chemoprotectant. In contrast, as described below, D-Met providescomplete otoprotection without apparent adverse side effects.

Finally, many sulfur-containing compounds inhibit the anti-tumor actionof CDDP, and it is not possible to predict which agents will or will notact in this manner. Thus, many agents that provide CDDP protection arenot clinically useful. For example, Captropril (Jones et al., 1992)protects against CDDP nephrotoxicity, but reacts immediately with CDDPto form a precipitate if coadministered, thereby precluding anti-tumorefficacy. L-methioninamide (Jones et al., 1991b) provides excellent CDDPnephroprotection, but impairs CDDP anti-tumor action. Metallothionein, asulfur-containing compound the synthesis of which is induced byadministration of bismuth subnitrate, provides CDDP nephroprotection,but also inhibits CDDP anti-tumor action (Naganuma et al., 1987;Boogaard et al., 1991; Satoh et al., 1993; Imura et al., 1992; Endresenet al., 1994). STS reduces CDDP nephrotoxicity (Pfeifle et al., 1985;Howell et al. 1982) and ototoxicity (Otto et al., 1988; Church et al.,1995), although some authors report inadequate otoprotection (Markman etal., 1985). However, STS will probably not be clinically useful ascoadministration with CDDP reduces the latter's tumoricidal action(Pfeile et al., 1985; Aamdal et al., 1987; Jones et al., 1991b), and tworoute administration does not provide nephroprotection (Jones et al.,1991b). Even in the absence of other agents, STS may also increasemortality and induce weight loss (Otto et al., 1988). Biotin, anothersulfur-containing compound that provides good CDDP nephroprotection,inhibits anti-tumor activity (Jones et al., 1992).

Thus, a variety of sulfur-containing compounds can act as protectiveagents for particular toxicities. A comparison of C—SH— andC—S—C-containing compounds demonstrated that the C—S—C— group was moreeffective in preventing nephrotoxicity in rats (Jones et al., 1989).However, not all of the compounds possessing the C—S—C— group were foundto be effective cisplatin antagonists.

The foregoing discussion demonstrates that it is not possible to predictreliably which particular sulfur-containing nucleophile will exhibit aplatinum-containing compound protective effect in any particular type ofcell, tissue, or organ. Indeed, individual compounds seem to exert theirprotective effects only in certain tissues. Thus, the ability of anyparticular nucleophilic sulfur compound to act as a protective agent inany particular tissue can only be determined by direct experimentation.Of course, such compound will only be of value if it does notsubstantially reduce the anti-tumor efficacy of cisplatin or relatedanti-tumor platinum-containing compounds.

Deegan et al. (1994) demonstrated that male Wistar rats receiving asingle intraperitoneal dose of cisplatin-methionine at a 1:5 ratio byweight did not exhibit cisplatin-induced nephrotoxicity. Their resultsindicated that cisplatin-methionine is significantly cytotoxic, yetlacks cisplatin-associated renal toxicity. These workers suggested arole for either methionine co-treatment or cisplatin-methioninecompounds in the treatment of human cancers. However, they neitherdisclosed nor suggested the specific otoprotective, weightloss-protective, gastrointestinal-protective, neuroprotective,alopecia-protective, or survival-enhancing effects of D-methioninesurprisingly discovered by the present inventor. Nor did they provideany motivation to investigate D-methionine as an otoprotectant, weightloss-protectant, survival-enhancing agent, etc., or any reasonableexpectation that methionine could act in these manners during cisplatinadministration. Finally, Deegan et al. provided no guidance orsuggestion as to how methionine could be used as a protective agent forvarious toxicities in humans, as described herein. As noted bySchweitzer (1993; page 12), while various nucleophilic sulfur protectiveagents have been shown to be effective in blocking or reversing therenal toxicity of CDDP while retaining the chemotherapeutic activity ofthe drug, each agent has to be considered individually. The effects onantineoplastic activity, individual CDDP toxicities, and appropriatedosing schedules need to be determined on a per se basis for eachcompound.

In view of the foregoing, the utility of D-Met as a highly effectiveplatinum-containing anti-neoplastic agent otoprotectant, weight lossprotectant, gastrointestinal protectant, neuroprotectant, alopeciaprotectant, and survival-enhancing agent which does not interfere withanti-tumor activity, and which does not appear to cause any serious sideeffects, could not have been predicted. In fact, the discovery ofD-Met's beneficial effects is surprising in view of the many significantproblems, discussed above, encountered with previously describedsulfur-containing nucleophiles that preclude their clinical use.

SUMMARY OF THE INVENTION

The present inventor has addressed the long-felt need in the art forprotective agents effective in preventing or ameliorating various toxiceffects of cisplatin and other platinum-containing anti-tumor compounds,but which do not significantly affect the antineoplastic activity ofthese compounds, and which do not themselves cause deleterious sideeffects as a result of their administration. She has surprisinglydiscovered that D-methionine, and structurally related compounds, can beused as an otoprotectant, a weight loss protectant, a gastrointestinalprotectant, a neuroprotectant, an alopecia protectant, and asurvival-enhancing agent during treatment of a mammal with such aplatinum-containing anti-tumor compound.

Accordingly, in one aspect, the present invention provides a method forpreventing or reducing ototoxicity in a human or animal patientundergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-ototoxic effective amount of a methionineprotective agent. Such protective agent can be administered prior to,simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In another aspect, the present invention provides a method forpreventing or reducing weight loss in a human or animal patientundergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-weight loss effective amount of a methionineprotective agent. Such protective agent can be administered prior to,simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In another aspect, the present invention provides a method forpreventing or reducing gastrointestinal toxicity in a human or animalpatient undergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-gastrointestinal toxicity effective amount of amethionine protective agent. Such protective agent can be administeredprior to, simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In yet another aspect, the present invention provides a method forpreventing or reducing neurotoxicity in a human or animal patientundergoing treatment with an anti-cancer effective amount of aplatinum-containing chemotherapeutic agent, comprising administering tosaid patient an anti-neurotoxicity effective amount of a methionineprotective agent. Such protective agent can be administered prior to,simultaneously with, or subsequently to administration of saidplatinum-containing chemotherapeutic agent. Combinations of these timeperiods can also be employed.

In still another aspect, the present invention provides a method forpreventing or reducing alopecia in a human or animal patient undergoingtreatment with an anti-cancer effective amount of a platinum-containingchemotherapeutic agent, comprising administering to said patient ananti-alopecia effective amount of a methionine protective agent. Suchprotective agent can be administered prior to, simultaneously with, orsubsequently to administration of said platinum-containingchemotherapeutic agent. Combinations of these time periods can also beemployed.

In yet another aspect, the present invention provides a method forprolonging the survival of a human or animal patient undergoingtreatment with an anti-cancer effective amount of a platinum-containingchemotherapeutic agent, comprising administering to said patient asurvival-prolonging effective amount of a methionine protective agent.Such protective agent can be administered prior to, simultaneously with,or subsequently to administration of said platinum-containingchemotherapeutic agent. Combinations of these time periods can also beemployed.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the following detailed descriptionand examples, while indicating preferred embodiments of the invention,are given by way of illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 shows ABR post-test thresholds (means ±1 S.D.) for the variousanimal groups for all stimuli including: a) clicks; b) 1000 Hztonebursts; c) 4000 Hz tonebursts; d) 8000 Hz tonebursts; and e) 14000Hz tonebursts. * indicates significantly different from the CDDP-treatedcontrols at the p≦0.01 level.

FIGS. 2A-2F are SEM photomicrographs of: A) middle turn of untreatedcontrol; B) middle turn of treated control (16 mg/kg CDDP); C) middleturn of animal administered 300 mg/kg D-Met prior to the 16 mg/kg CDDPdose; D) basal turn of untreated control; E) basal turn of treatedcontrol (16 mg/kg CDDP); and F) basal turn of animal administered 300mg/kg D-Met prior to the 16 mg/kg CDDP dose.

FIG. 3 shows the average weight loss in grams for the various animalgroups. * indicates significantly different from the CDDP-treatedcontrols at the p≦0.01 level.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. Even so, this detaileddescription should not be construed to unduly limit the presentinvention as modifications and variations in the the embodimentsdiscussed herein can be made by those of ordinary skill in the artwithout departing from the spirit or scope of the present inventivediscovery.

The contents of each of the references cited herein are hereinincorporated by reference in their entirety.

EXAMPLE 1 Otoprotective Effect of D-Met

This experiment demonstrates the effectiveness of D-Met in preventing avariety of different toxic side effects associated with the use ofplatinum-containing anti-tumor compounds, exemplified by CDDP(cisplatin), in a mammal.

Materials and Methods

Animals

As is well known to those of ordinary skill in the art, the rat is awell-accepted experimental animal useful as a model for studies of CDDPtoxicity in humans.

Complete data sets were obtained for five groups of five male Wistarrats (280-421 g). All animals were anesthetized with 1 ml/mg IM ofRompun cocktail (a solution containing 86.21 mg/ml ketamine and 2.76mg/ml xylazine) prior to all injections and testing. Anesthesia wassupplemented as needed with half doses throughout testing. The fivegroups included: a treated control group which received 16 mg/kg CDDPdissolved in normal sterile saline (1 mg of CDDP/ml normal saline;solution pH 6.3) administered by i.p. infusion with a Harvard ApparatusInfusion Pump, over a 30 minute period, an untreated control group thatreceived an equivalent volume of normal saline (pH 6.5) instead of CDDP,and three experimental groups that received either 75, 150, or 300 mg/kgD-Met dissolved in 3-5 ml of normal saline (solution pH 6.6) deliveredby slow (over 1-2 minutes) i.p. injection 30 minutes prior to the sameCDDP infusion as the treated control group. Both CDDP (purchased fromSigma Chemical Co., St. Louis) and D-Met (puchased from Acros Organics,Pittsburgh, Pa.) were freshly prepared before each experiment. For thetreated control group, a total of 10 animals were needed to obtain 5animals with complete data sets because 50% of the animals did notsurvive to the end of the study period. Only 5 animals were needed inthe untreated control and in each of the D-Met pretreated groups becauseall of the animals in each of those groups survived until the end of thestudy period.

All of the care and use of the animals was approved by the SouthernIllinois University School of Medicine Laboratory Animal Care and UseCommittee, and was under the supervision of the Southern IllinoisUniversity School of Medicine Unit for Laboratory Animal Medicine.

Evoked Potentials

Auditory Brainstem Testing (ABR) was used to assess auditory threshold.Testing occurred just prior to administration of the CDDP or saline(with or without a protective agent) and again 3 days later. All testingwas performed with the animal in a double walled IAC booth.

Platinum/iridium needle electrodes were placed at the vertex(non-inverting) to a point directly below the ipsilateral pinna(inverting) with a ground electrode placed in the hind leg.

ABR data collection was obtained with a Biologic Traveler system with anadditional custom made high frequency stimulator for 14000 Hz. ABRthresholds were measured in response to 100 microsecond clicks and fortonebursts with 1 ms rise/fall and 0 ms plateau gated by a Blackmanenvelope and centered at the frequencies of 1, 4, 8, and 14 kHzpresented at 10/s. An intensity series was obtained for each animal from100 to 0 dB peak equivalent SPL (peSPL) for click stimuli and SoundPressure Level (SPL) for tonebursts in 10 dB decrements. The term peSPLmeans that the amplitude of the click stimulus from the prestimulusbaseline to the first peak is equivalent to the SPL of a pure tonestimulus having the same prestimulus baseline to peak amplitude.Threshold was defined as the lowest intensity capable of eliciting areplicable, visually detectable response.

A total of 512 sweeps constituted each average. The recording epoch was15 ms following stimulus onset. Responses were analogue filtered with a30-3000 Hz bandpass.

Rectal temperature was monitored throughout recordings, with animaltemperature being maintained by a warming pad.

Electron Microscopy

The animals were sacrificed by decapitation while under generalanesthesia and cochleae perfused with fixative through the perilymphaticspaces. The primary fixative was 2.5% glutaraldehyde at 4° C. in 0.1Mphosphate buffer (pH 7.4). A small hole in the otic capsule was handdrilled beneath the first turn with a three sided, sharpened pick. Invitro perfusion was performed intermittently within 5 minutes ofsacrifice through the small hole in scala tympani, allowing the fluid toexit through the opened oval window. After perfusion fixation, the roundwindow membrane was removed, and the cochleae were immersed inglutaraldehyde and stored in the refrigerator overnight.

After overnight fixation in glutaraldehyde, the cochleae were rinsed in0.1 M phosphate buffer and gently perfused with the buffer through theperilymphatic spaces by loosely fitting the tube end of the perfusionsyringe over the opening drilled in the scala tympani. Cochleae werethen rinsed in buffer 3 times. After rinsing, the cochleae werepost-fixed by a perfusion of 1.5% OsO₄ (at 4° C.) in phosphate buffer ina fume hood. Fixation was continued by immersion and rotation in thesame fixative for 15 minutes. The cochleae were rinsed in the samefashion as after glutaraldehyde fixation.

Under the dissecting microscope, the bony capsule of the cochlea wascarefully removed.

The tissue was then serially dehydrated in 2×50%, 70%, 85%, 95% and3×100% ethanol. Each specimen was dried using Peldri and placed on astub for sputter coating with 13 nm platinum. The tissue was viewedthrough a Hitachi S-500 scanning electron microscope and photographstaken on Polaroid type 55 land Film.

Semi-quantitative analysis per turn for the outer hair cells wasperformed in the following manner: For each turn of the cochlea, apical,middle, and base, a representative sample was examined. For each sample,11 inner hair cells served as a guide to count a section of 33 outerhair cells or 11 per row. The number of damaged or missing outer haircells within each sample was then counted.

Weight

Each animal's weight was measured in an Ohaus triple beam balance scalebefore administration of the anesthetic for the pretest and again beforethe post-test 3 days later.

Statistical Analysis

ABR data were analyzed using a three factor analysis of variance (ANOVA)with one between subject factor (groups) and two within subject factors(frequency and pre- vs. post-test). Each dependent variable was analyzedindependently. Tests subsequent to the ANOVA were carried out inaccordance with the Tukey HSD procedure. Weight loss and/orgastrointestinal protection was measured using the same type ofstatistical analysis as the ABR measures. SEM data were analyzed foreach turn using a one way analysis of variance with Post-Hoc Tukey HSDanalysis. The criterion for statistical significance for all measureswas p≦0.01.

Results

Hearing Loss

Post test ABR hearing thresholds are presented in FIG. 1. As expected,no significant threshold shift in response to any stimulus occurred inthe untreated control group, and marked significant threshold shiftoccurred in response to all stimuli, but particularly for the highfrequencies, in the treated control group. For the animals receivingD-Met prior to the CDDP, 2/5 and 3/5 animals receiving 75 and 150 mg/kgD-Met, respectively, had complete otoprotection as defined by nosignificant ABR threshold shift for any stimulus. For the 300 mg/kgD-Met administration, all 5 animals had complete otoprotection for allstimulus conditions (FIG. 1). All experimental groups receiving anylevel of D-Met had significantly lower ABR thresholds than the treatedcontrol group for all stimuli, as did the untreated control group. Thisobserved protection from hearing loss may occur not only as a result ofprotection of cochlear mechanisms, but also as a result of protection ofthe auditory neural pathway (i.e., neuroprotection).

Histology

Histologic findings (FIG. 2) were consistent with the ABR findings. Allgroups had essentially normal hair cell counts for the apical turn, withno significant difference between groups. For the middle and basalturns, only the treated control group showed significantly differentfindings from the untreated control group and from the three groupsreceiving preadministration of D-Met, with the basal turn beingconsistently more affected than the middle turn.

Weight loss

CDDP-induced weight loss diminished as D-Met dosing increased (FIG. 3).Weight loss in the experimental group receiving 300 mg/kg wassignificantly less than that in the treated control group. The amount ofweight loss across groups was significantly correlated with the amountof threshold shift for all stimuli, with the highest correlation for the14 kHz stimulus.

Neuroprotection

Animals receiving D-Met were noticeably more lively, active, andcoordinated on the morning of the third day as compared to the survivingtreated control group animals.

Alopecia

The coats of animals receiving D-Met were noticeably superior to thoseof control group animals, and showed significantly less hair loss.

Survival During the Study Period

All 15/15 animals receiving any level of D-Met survived to the end ofthe study period as compared to 5/10 treated control group animals.

Discussion

The foregoing results demonstrate that 300 mg/kg D-Met administered 30minutes before 16 mg/kg CDDP provides complete otoprotection, asindicated by ABR and histologic findings, while also reducingCDDP-induced weight loss, gastrointestinal toxicity, neurotoxicity,alopecia, and improving survival.

While not intending to be bound to any particular theory, I hypothesizethat D-Met may provide these protective effects by any one or more of anumber of different mechanisms.

According to Schweitzer, (1993), sulfur-containing compounds may preventCDDP from interacting with intracellular target molecules, thenucleophilic oxygen or sulfur atoms interacting with the electrophilicsite of the CDDP, thus displacing or extracting platinum after it isbound. Theoretically, these agents provide protection because of theirhigh affinity for platinum complexes. It is known that CDDP reacts withmethionine's sulfhydryl group (Lempers and Reedijk, 1990).

CDDP may preferentially bind to free D-Met, thus protecting glutathione.Reduced glutathione is an essential part of the anti-oxidant pathways.CDDP does reduce renal glutathione levels, resulting in increased lipidperoxidation (Hanneman and Baumann, 1988; Sugihara et al., 1987a, b;Boogaard, 1991). CDDP also reduces glutathione levels in the cochlea andinferior colliculus (Ravi et al., 1991). More recent work (Ravi et al.,1995, Rybak et al., 1995) investigated changes specifically in thecochlear antioxidant system. Systemic CDDP administration decreasedreduced glutathione (GSH) levels, and reduced activity of the enzymesglutathione peroxidase (GSH-Px) and glutathione reductase (GR). Oxidizedglutathione or glutathione disulfide (GSSG) was not found, suggestingthat the overall glutathione levels decreased rather than merely beingoxidized. Ravi et al., (1995) also reported increased cochlearmalondialdehyde (MDA) levels, reflecting increased lipid peroxidation.Because CDDP does increase the level of free radicals in general(Hanneman and Baumann, 1988), preservation of the anti-oxidant systemmay be critical in preventing CDDP side effects.

D-Met preadministration may protect the sulfur groups of proteins,including protein bound L-methionine. CDDP binds to the methioninegroups in protein and to glutathione (Lempers and Reedijk, 1990).Schweitzer, (1993) suggests that platinum binding to protein sulfhydrylgroups may cause CDDP nephrotoxicity, accounting for thenephroprotective action of thiols (Gandara et al., 1989). It is logicalthat free D-Met may preferentially bind to CDDP because of the sterichindrance of the protein bound sulfur groups. This protection couldoccur by preferential binding of the CDDP to D-Met, or perhaps D-Metcould reverse the Pt binding to the protein-bound methionine andglutathione, as do other sulfur-containing compounds (Lempers andReedijk, 1990). Methionine can displace plasma-bound Pt (Alden andRepta, 1984).

D-Met binding to CDDP may also protect free L-methionine (L-Met), anessential amino acid. Parenteral administration of DL-methionine inhumans results in higher plasma levels of the D-isomer (Printen et al.,1979). Because the D-Met is less well metabolized than L-Met in humans,it may remain more available for CDDP binding, thus protecting the L-Metfor needed protein synthesis, cell activation, and metabolism.

Fortunately, D-Met does not inhibit CDDP anti-tumor action as determinedagainst the Walker 256 carcinosarcoma in the rat (Jones and Basinger,1989). Preadministration of methionine, presumably a racemic mixture,actually sensitized NHIK 3025 in vitro human uterine cervix carcinoma insitu cancer cells to CDDP cytotoxicity (Melvik and Petterson, 1987).

Several factors may account for D-Met's CDDP-protective action innontumor cells as compared to tumor cells. Methionine metabolism isclearly different in tumor and nontumor cells (Hoffman, 1985), but howthese differences may result in differential CDDP action has not beenelucidated. The toxic effects of CDDP may also be different in tumor andnontumor cells. The CDDP anti-tumor effect results primarily fromcisplatin's reaction with DNA, primarily at the N-7 bisguanine position.Initially, mono-adducts are formed, followed by rapid intra-strandcross-linking, causing cytotoxicity (see the review by Tognella, 1990).The binding of platinum to cytosolic ligands and nucleoprotein fractionsmay also play a role, but the receptors and interactions are not yetdefined (Schweitzer, 1993). Significant DNA binding in normal cells isless likely because fewer DNA replication forks are open at any point intime, unlike in rapidly dividing tumor cells. In nontumor cells, thetoxic effects may be largely secondary to the binding with amino acids,either free or protein-bound, and deactivation of the antioxidantpathway, as described above.

The timing of CDDP reactions may also be different in tumor and nontumorcells. CDDP uptake by the Walker 256 carcinosarcoma in the rat is veryrapid, occurring in the first few minutes after administration, followedby a rapid redistribution that is complete within 15 minutes afterinjection (Jones and Basinger, 1989). Because the uptake of CDDP intotumor cells is very rapid, the binding to the DNA bisguanine groups,particularly at the open replication forks, may occur more rapidly thanthe reaction of CDDP with methionine.

Although CDDP uptake into the kidney is also rapid (Jones and Basinger,1989), CDDP binding to protein is relatively slow. As reviewed bySchweitzer, (1993), following IV cisplatin administration, 90% ofcisplatin is protein-bound within 2 hours, with half-lives of 25 to 50minutes and 53 to 73 hours for unbound and bound platinum, respectively.Platinum tissue levels decline slowly. Platinum may still be measuredover a week after high dosage administration, and bound fragments maystill be present when the patient starts the next treatment cycle.Platinum uptake in the stria vascularis and the organ of Corti increasesat least over a 24 hour period, which may underlie the dose-relatedcumulative ototoxicity (Schweitzer, 1993), but may also allow time forCDDP binding to D-Met before uptake into the cochlea.

However, the CDDP toxicities both in tumor and nontumor cells arecomplex, and many factors may be involved in D-Met's protective action.

A positive correlation between weight loss and outer hair cell loss inguinea pigs has been demonstrated (Tange et al., 1982, Hoeve et al.,1988), but both studies noted marked intersubject variability. The datapresented above reveal a positive correlation between weight loss andthreshold loss that increased as stimulus frequency increased. Thesignificant reduction in weight loss with 300 mg/kg D-Metpreadministration suggests that D-Met also alleviates some of thegastrointestinal toxicities of CDDP. The amelioration in weight loss byD-Met could also be related to a decrease in nephrotoxicity or otherfactors.

The elimination of CDDP mortality in this study by preadminstration ofany of the three D-Met levels demonstrates a marked improvement in theoverall health status of the animals. D-Met preadministration maytherefore be useful in shifting the LD₅₀ level of CDDP and otherplatinum-containing anti-tumor agents, permitting the safe use of higherlevels of these agents during chemotherapy, with potential improvementof the cancer cure rate.

Therapeutic Applications

The data presented above demonstrate that D-Met prevents CDDP-inducedototoxicity, reduces CDDP-induced weight loss, protects againstCDDP-induced gastrointestinal toxicity, neurotoxicity, and alopecia, andimproves survival during CDDP treatment in a mammal. BecauseCDDP-induced hearing loss in humans is almost invariably permanent,prevention of this hearing loss has a number of important consequences.If ototoxicity could be prevented, not only could hearing be spared, butperhaps higher doses of cisplatin and other platinum-containingantineoplastic agents could be routinely employed during chemotherapy,increasing the effectiveness of anti-tumor therapy in human patients.

As used herein, the term “ototoxicity” includes, but is not limited to,any detrimental or pathologic change in the structure or function of theear, including changes in hearing and balance. Auditory functionalchanges can include, but are not limited to, hearing loss or otherchanges in auditory threshold for any stimulus, perception of soundincluding recruitment (abnormal growth in the perception of loudness),ability to identify, localize, recognize, distinguish between, orprocess sounds, and/or distortion of sounds or any abnormality asmeasured by conventional auditory tests. This term also includestinnitus (ringing or noises in the ear), which includes any perceptionof sound that is not in response to an external signal. Further,ototoxicity includes any perceived or measured functional change in thebalance or vestibular system, including, but not limited to, eitherinduced or spontaneous vertigo, dysequilibrium, increased susceptibilityto motion sickness, nausea, vomiting, nystagmus, syncope,lightheadedness, dizziness, difficulty in visual tracking secondary tovestibular or balance disorder or abnormality as measured on any test ofvestibular or balance function. Structural changes can include anyintra- or extra-cellular, multicellular, or organ change in the auditoryor vestibular pathways from the external ear up through and includingthe cortex and all pathways in between.

The term “otoprotective agent” refers to an agent that prevents,ameliorates, or otherwise protects against otoxicity.

The term “neurotoxicity” includes, but is not limited to, anydetrimental or pathologic change in the structure or function in theneurologic system or any part thereof. Neurologic functional changes caninclude, but are not limited to, neuropathy, either central or distal,including a common “stocking and glove” pattern, tingling, loss ofsensation, numbness, decreased vibratory sensation, decreased deeptendon reflexes, sensory ataxia, neuritis, focal encephalopathy,aphasia, autonomic neuropathy, postural hypotension, a myasthenia-likesyndrome, muscle cramps, headache, seizures, blindness or visualdisturbance secondary to disorder of the optic or visual neurologicalpathway, papilledema, hearing loss secondary to disorder of the auditoryneurologic pathway, and/or loss of the sensation of taste. Structuralchanges can include intra- or extra-cellular, multicellular, or organchanges, anywhere in the neurologic system, including both peripheraland central systems. Neurotoxicity can manifest itself during or afterthe course of treatment with platinum-containing anti-tumor compounds.

The term “neuroprotective agent” refers to an agent that prevents,ameliorates, or otherwise protects against neurotoxicity.

The term “gastrointestinal toxicity” includes, but is not limited to,any detrimental or pathologic change in the structure or function in thegastrointestinal system or any part thereof. Gastrointestinal changesinclude, for example, current or delayed nausea, vomiting, esophagealreflux, stomatitis, bleeding along the gastrointestinal tract, diarrhea,weight loss, and/or anorexia. Gastrointestinal toxicity can manifestitself during or after the course of treatment with platinum-containinganti-tumor compounds.

The term “gastrointestinal-protective agent” refers to an agent thatprevents, ameliorates, or otherwise protects against gastrointestinaltoxicity.

In view of the results presented above, the medical or veterinarypractitioner, by employing the compounds and methods described below,will be able to maintain any of the foregoing parameters in a mammal,especially a human, at a level of from about 70% to about 80% of thepre-chemotherapy treatment level, more preferably from about 80% toabout 90% of the pre-chemotherapy treatment level, most preferably fromabout 90% to about 100% of the pre-chemotherapy treatment level, asmeasured by standard tests routinely employed in the art. Thesecompounds and methods can also be used for the treatment of domesticpets, such as cats and dogs.

The teachings presented herein permit the design of therapeutic regimensthat can be employed to reduce the undesirable side effects ofplatinum-containing anti-tumor compounds such as CDDP, increase thedosing of such anti-tumor compounds to obtain a higher cancer cure rate,and perhaps include weaker patients in treatment protocols employingsuch anti-tumor compounds, from which they are currently excludedbecause they cannot withstand the toxicities associated therewith.Administration of D-Met before, during, or after administration ofantineoplastic effective amounts of platinum-containing anti-tumorcompounds such as CDDP, or during various combinations of these timeperiods, is particularly useful in view of D-Met's lack of interferencewith CDDP anti-tumor action (Jones and Basinger, 1989; Melvik andPetterson, 1987).

D-Met and structurally related compounds can be used in conjunction withplatinum-containing antitumor compounds such as CDDP during chemotherapyas described below.

Methionine and its Derivatives

D-Met has been administered to humans for various purposes. For example,C-labeled D-Met has been used for radiographic imaging (Meyer et al.,1985), and DL-methionine has been administered for parenteral nutrition(Printen et al., 1979). D-Met has also been safely administered tohumans orally for nutritional studies (Kaji et al., 1987; Kies et al.,1975; Stegink et al., 1986). Oral methionine is sold as an over thecounter preparation to control urinary pH (Drug Facts and Comparisons,1991). The contraindications are for patients with a history of liverdisease, and that high dosage methionine may inhibit growth in childrenwhen given for an extended time period.

Analogs or derivatives of methionine useful in the present invention arecompounds containing a methionine moiety, or a methionine-like moietyincluding a thioether group, that exhibit an otoprotectant effect, aweight-loss protectant effect, a gastrointestinal protectant effect, aneuroprotectant effect, an alopecia protectant effect, and/or asurvival-enhancing effect when used in conjunction with an antitumorplatinum coordination compound administered in an effectivechemotherapeutic dose. Among the compounds structurally related to D-Metthat can be employed in the present invention are those containing theC—S—C— (thioether) moiety. These include, but are not limited to,compounds having the structural formula:

wherein m is an integer from 0 to 3; n is an integer from 1 to 3;X=—OR¹, —OCOR¹, —COOR¹, —CHO, —CH(OR¹)₂, or —CH₂OH; Y=−NR²R³ or —OH;R¹=H or a substituted or unsubstituted, straight, branched chain, orcyclic alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms; R²=H or a substituted or unsubstituted, straight or branchedchain acyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms; and R³=H or a substituted or unsubstituted, straight or branchedchain acyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbonatoms; or a pharmaceutically acceptable salt thereof.

The lower alkyl and acyl groups described herein, either alone orcontaining the various substituents defined herein, can contain from oneto six carbon atoms in the principal chain, and up to about 15 carbonatoms total. The lower alkyl groups include, for example, methyl, ethyl,propyl, isopropyl, butyl, hexyl, cyclopropyl, cyclopentyl, cyclohexyl,and the like. Substituents of the substituted alkyl and acyl groupsdescribed herein can include, for example, groups selected from alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, O, S, N, P, or halogen(Cl, F, Br, or I) atoms. Optionally, these substituent aklyl,cycloalkyl, etc., groups can be substituted with O, S, N, P, or halogen(Cl, F, Br, or I) atoms. These substituent alkyl, cycloalkyl, etc.,groups include, for example, lower alkoxy groups such as methoxy,ethoxy, and butoxy, and groups such as halo, nitro, amino, and keto.

The alkenyl groups described herein, either alone or with the varioussubstituents defined herein, are preferably lower alkenyl containingfrom two to six carbon atoms in the principal chain, and up to about 15carbon atoms total. They can be substituted, straight, or branchedchain, and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl,hexenyl, and the like.

The alkynyl groups described herein, either alone or with the varioussubstituents defined herein, are preferably lower alkynyl containingfrom two to six carbon atoms in the principal chain, and up to about 15carbon atoms total. They can be substituted, straight or branched chain,and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and thelike.

The aryl moieties described herein, either alone or with varioussubstituents defined herein, can contain from about 6 to about 15 carbonatoms, and include phenyl. Substituents include alkanoxy, protectedhydroxy, halogen, alkyl, aryl, alkenyl, acyl, acyloxy, nitro, amino,amido, etc. Phenyl is a preferred aryl.

The heteroaryl moieties described herein, either alone or with varioussubstituents defined herein, can contain from about 5 to about 15 atoms,and include, furyl, thienyl, pyridyl and the like. Substituents includealkanoxy, protected hydroxy, halogen, alkyl, aryl, alkenyl, acyl,acyloxy, nitro, amino, and amido.

The acyloxy groups described herein can contain alkyl, cycloakyl,alkenyl, alkynyl, aryl, or heteroaryl groups.

The carbon atoms, i.e., the methyl and methylene groups, constitutingthe principal backbone of the methionine or methionine-like moiety canalso be substituted as variously described above.

Non-limiting examples of such methionine protective agents includeD-methionine (D-Met), L-methionine, a mixture of D-methionine andL-methionine, normethionine, homomethionine, methioninol, hydroxymethionine, ethionine, or pharmaceutically acceptable salts thereof.Methionine protective agents of the present invention can be in the D-,L-, or DL- form, and include pharmaceutically acceptable N-(mono- anddicarboxylic acid) acyl derivatives and alkyl esters thereof. Exemplaryacyl derivatives include the formyl, acetyl, propionyl, and succinylderivatives. Exemplary ester derivatives include the methyl, ethyl,propyl, isopropyl, and butyl esters. D-Met is a preferred compound.

Collectively, methionine, along with the other compounds discussedabove, can be referred to as “methionine protective agents.” Thesecompounds can be used alone or in various combinations with one anotherin the methods described herein.

These compounds can be administered in the form of the water-solubleacid, free base, or as physiologically acceptable salts, including acidaddition salts formed with organic and inorganic acids, for example,hydrochlorides, hydrobromides, sulfates, phosphates, citrates,fumarates, and maleates, and cations such as sodium, potassium, etc.These compounds can be formulated for administration to humans andanimals with pharmaceutically acceptable carriers, excipients, anddiluents, such as sterile distilled water, Ringer's solution, normalsaline, 5% glucose, dextrose, fructose, sucrose, etc., and mixturesthereof, as is well known in the art. Antimicrobial agents,preservatives, etc., can also be included. Compositions for oraladministration can include coloring and flavoring agents. Additionalmethods of formulating compounds of the present invention foradministration in the methods described herein can be found, forexample, in Remington's Pharmaceutical Sciences, Fifteenth Edition, MackPublishing Company, Easton, Pa., 1975.

Anti-tumor Platinum Compounds

Cisplatin (CDDP; cis-diamminedichloro-platinum(II)) is currently theanti-tumor platinum coordination compound most frequently employed inthe therapy of testicular cancer, ovarian tumors, and a variety of othercancers. Methods of employing CDDP clinically are well known in the art(Nicolini, 1987). For example, CDDP can be administered in a single dayover a six hour period, once per month, by slow intravenous infusion.For localized lesions, CDDP can be administered by local injection.Intraperitoneal infusion can also be employed. CDDP can be administeredin doses as low as 10 mg/m² per treatment if part of a multi-drugregimen, or if the patient has an adverse reaction to higher dosing. Atthe low end, a more common clinical dose is about 30 mg/m²; the high endof the range is about 120 to about 150 mg/m² per treatment. When used inconjunction with D-Met or other methionine protective agents, thesedosages can be increased.

CDDP is representative of a broad class of water-soluble, platinumcoordination compounds well known in the art that provide platinum inthe form of an ion having anti-tumor activity. Among the anti-tumorplatinum coordination compounds described in the literature which areuseful in the methods of the present invention are, for example,trans-diaminedichloro-platinum(II), cis-diamine-diaquaplatinum(II)-ion,cis-diaminedichloroplatinum(II)-ion,chloro(diethylenetriamine)-platinum(II) chloride,dichloro(ethylenediamine)-platinum(II),diammine(1,1-cyclobutanedicarboxylato)-platinum(II) (carboplatin),spiroplatin, dichlorotrans-dihydroxybisisopropolamine platinum IV(iproplatin), diammine(2-ethylmalonato)platinum(II),ethylenediamine-malonatoplatinum(II),aqua(1,2-diaminodiclohexane)-sulfatoplatinum(II),(1,2-diaminocyclohexane)malonato-platinum(II),(4-carboxyphthalato)(1,2-diaminocyclo-hexane)-platinum(II),(1,2-diaminocyclohexane)-(isocitrato)platinum(II),(1,2-diaminocyclohexane)-cis(pyruvato)platinum(II), and(1,2-diaminocyclohexane)-oxalatoplatinum(II).

Administration of Methionine Protective Agents

The methionine protective agents of the present invention can beadministered orally or parenterally, for example intraperitoneally, byintravenous injection, intravenous infusion, etc., as described inRemington's Pharmaceutical Sciences, Fifteenth Edition, Mack PublishingCompany, Easton, Pa., 1975. These protective agents can also be given bylocal administration when the platinum-containing chemotherapeutic agentis administered by local injection, as noted above. Localizedadministration of methionine protective agents can be carried out bytopical application employing pharmaceutical formulations designed forthis purpose as is known in the art, local injection, etc.

Administration of the methionine protective agents of the presentinvention simultaneously with the administration of aplatinum-containing chemotherapeutic agent can be accomplished inseveral ways. For example, each can be formulated individually andadministered separately at the same time via any of the routes describedherein. Alternatively, both can be contained together in a single dosageformulation that can be administered by a single route. As in the caseof the platinum-containing chemotherapeutic agent, the dose ofmethionine protective agent can be administered in a single day.

Dosages

The methionine protective agents discussed above can be employed inmethods for treating human and animal patients undergoing treatment withanti-cancer effective amounts of platinum-containing chemotherapeuticagents to prevent or reduce ototoxicity, weight loss, gastrointestinaltoxicity, neurotoxicity, alopecia, and to prolong survival. Thesemethods comprise administering to the patient an appropriate effectiveamount of a methionine protective agent prior to, simultaneously with,or subsequent to administration of the platinum-containingchemotherapeutic agent. Combinations of these time periods can also beemployed.

When administered parenterally, the effective amount of methionineprotective agent can be in the range of from about 0.1 mg/kg body weightto about 500 mg/kg body weight, more preferably from about 1 mg/kg bodyweight to about 400 mg/kg body weight, even more preferably from about10 mg/kg body weight to about 300 mg/kg body weight, and still morepreferably from about 1 mg/kg body weight to about 100 mg/kg bodyweight. A preferred amount is from about 10 mg/kg body weight to about75 mg/kg body weight.

Alternatively, the effective amount of methionine protective agent canbe expressed on a mole:mole basis in relation to the anti-cancereffective amount of platinum-containing chemotherapeutic agent. Thiseffective amount can be in the range of from about 4:1 to about 167:1,more preferably from about 4.25:1 to about 100:1, and most preferablyfrom about 4.68:1 to about 20:1, methionine protectiveagent:platinum-containing chemotherapeutic agent, on a molar basis. Adosing ratio of about 18.75:1 on a molar basis is a preferred ratio. Ifnecessary, these ratios can be modified for differentplatinum-containing chemotherapeutic agents by routine optimization,including monitoring of effectiveness and titration for the desiredeffect, by the methods described herein.

When administered orally, the methionine protective agent should begiven in an amount that will result in a blood serum level equivalent tothat achieved by the parenterally administered doses set forth above.Such effective oral doses can easily be determined by one of ordinaryskill in the art via conventional in vitro or in vivo methods such asthose described in Remington's Pharmaceutical Sciences, FifteenthEdition, Mack Publishing Company, Easton, Pa., 1975.

Treatment Regimen

In the various methods of the present invention, the effective amount ofsulfur-containing protective agent can be administered prior to,contemporaneously with, or subsequent to administration of the effectiveamount of platinum-containing chemotherapeutic agent. Combinations ofthese time periods can also be employed. Prior administration of theeffective amount of the methionine protective agent can be within about36 hours before administration of the platinum-containingchemotherapeutic agent; subsequent administration can be within about 36hours after administration of the platinum-containing chemotherapeuticagent. More preferably, prior administration can be within about 25hours before, and subsequent administration can be within about 25 hoursafter, administration of the platinum-containing chemotherapeutic agent.More preferably, prior administration can be within about 6 hoursbefore, and subsequent administration can be within about 1 hour after,administration of the platinum-containing chemotherapeutic agent. Evenmore preferably, prior administration of the effective amount ofmethionine protective agent can be within about 1 hour before, andsubsequent administration can be within about 1 hour after,administration of the platinum-containing chemotherapeutic agent. Stillmore preferably, prior administration of the effective amount ofmethionine protective agent can be within about one-half hour before,and subsequent administration can be within about one-half hour after,administration of the platinum-containing chemotherapeutic agent.

The platinum-containing chemotherapeutic agent can be administeredparenterally, for example by slow intravenous infusion, or by localinjection, as discussed above. The methionine protective agent can beadministered orally, or parenterally by intravenous injection or slowinfusion, or intraperitoneally.

Delayed toxic effects due to platinum-containing chemotherapeutic agentshave been observed. The protective effects of the present methionineprotective agents can be enhanced by administering them in asupplemental manner during the course of the patient's chemotherapyand/or afterwards as necessary or as desired. Thus, the methodsdescribed herein can further comprise daily or weekly parenteraladministration of a supplemental amount of methionine protective agentin the range of from about 0.1 mg/kg body weight to about 500 mg/kg bodyweight, more preferably from about 1 mg/kg body weight to about 400mg/kg body weight, even more preferably from about 10 mg/kg body weightto about 300 mg/kg body weight, and still more preferably from about 1mg/kg body weight to about 100 mg/kg body weight. A preferred amount isfrom about 10 mg/kg body weight to about 75 mg/kg body weight.

Alternatively, the effective amount of methionine protective agentparenterally administered daily or weekly can be expressed on amole:mole basis in relation to the anti-cancer effective amount ofplatinum-containing chemotherapeutic agent. This effective amount can bein the range of from about 4:1 to about 167:1, more preferably fromabout 4.25:1 to about 100:1, and most preferably from about 4.68:1 toabout 20:1, methionine protective agent:platinum-containingchemotherapeutic agent, on a molar basis. A dosing ratio of about18.75:1 on a molar basis is preferred.

Oral or parenteral doses administered daily can be within the lowerranges listed above. When administered orally, daily or weekly dosesshould be designed to achieve serum levels equivalent to those achievedby administration of the various parenteral doses described above.

The supplemental methionine protective agent can be administeredparenterally by intravenous injection or slow infusion, orintraperitoneally.

Optimization of Treatment Regimen

In the method of preventing or reducing ototoxicity of the presentinvention, various parameters associated with the patient's hearing andvestibular systems can be tested by methods well known in the art toestablish pretreatment baseline values. After administration of themethionine protective agent, and over the course of chemotherapy andafterwards, ototoxic effects can be monitored by conventional tests, andthe results can be compared to those obtained prior to treatment todetermine if any change has occurred. If any impairment is observed, theamount and/or time of administration of the protective agentadministered in conjunction with subsequent doses of theplatinum-containing chemotherapeutic agent can be adjusted so as toreduce or prevent further ototoxic changes without substantiallydiminishing the antineoplastic effectiveness of the platinum-containingchemotherapeutic agent. Similar modification of treatment parameters inthe case of weight loss, gastrointestinal toxicity, neurotoxicity,alopecia, and overall patient condition/survival can be employed tooptimize the protective effects of the protective agent with respectthereto. This can be achieved via appropriate testing and comparison ofpre- and post-treatment values, e.g., patient weight and patientphysical/medical/physiological condition, etc., with protocoladjustments being made as needed.

The invention being thus described, it will be obvious that the same canbe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications and equivalents as would be obvious to one skilled inthe art are intended to be included within the scope of the followingclaims.

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What is claimed is:
 1. A method for preventing or reducing ototoxicityin a patient selected from the group consisting of a human, a cat, and adog undergoing treatment with a chemotherapeutic effective amount of ananti-tumor platinum-coordination compound, comprising administering tosaid patient an anti-ototoxic effective amount of D-methionine, whereinthe D-methionine is substantially free of the L-isomer.
 2. The method ofclaim 1, wherein said D-methionine is administered prior toadministration of said platinum-containing chemotherapeutic agent. 3.The method of claim 1, wherein said D-methionine is administeredsimultaneously with administration of said anti-tumorplatinum-coordination compound.
 4. The method of claim 1, wherein saidD-methionine is administered subsequently to administration of saidanti-tumor platinum-coordination compound.
 5. The method of claim 1,wherein said effective amount of said D-methionine is administered tosaid patient in a time period from about 36 hours before administrationof said anti-tumor platinum-coordination compound to about 36 hoursafter administration of said anti-tumor platinum-coordination compound.6. The method of claim 1, wherein said effective amount of saidD-methionine is administered to said patient in a time period from about25 hours before administration of said anti-tumor platinum-coordinationcompound to about 25 hours after administration of said anti-tumorplatinum-coordination compound.
 7. The method of claim 1, wherein saideffective amount of said D-methionine is administered to said patient ina time period from about 6 hours before administration of saidanti-tumor platinum-coordination compound to about 6 hours afteradministration of said anti-tumor platinum-coordination compound.
 8. Themethod of claim 1, wherein said effective amount of said D-methionine isadministered to said patient in a time period from about 1 hour beforeadministration of said anti-tumor platinum-coordination compound toabout 1 hour after administration of said anti-tumorplatinum-coordination compound.
 9. The method of claim 1, wherein saideffective amount of said D-methionine is administered to said patient ina time period from about one-half hour before administration of saidanti-tumor platinum-coordination compound to about one-half hour afteradministration of said anti-tumor platinum-coordination compound. 10.The method of claim 1, wherein said anti-tumor platinum-coordinatorcompound is selected from the group consisting ofcis-diamminedi-chloroplatinum(II), trans-diaminidichloroplatinum(II),cis-diammine-diaquaplatinum(II)-ion,chloro(diethylenetriamine)-platinum(II) chloride,dichloro(ethylene-diamine)-platinum(II),diammine(1,1-cyclobutanedi-carboxylato)-platinum(II), spiroplatin,dichlorotrans-dihydroxybisisopropolamine platinum IV (iproplatin),diammine(2-ethylmalonato)-platinum(II),ethylenediamine-malonatoplatinum(II),aqua(1,2-diaminodyclohexane)-sulfatoplatinum(II),(1,2-diaminocyclohexane)malonato-platinum(II),(4-carboxyphthalato)(1,2-diaminocyclo-hexane)-platinum(II),(1,2-diaminocyclohexane)-(isocitrato)platinum(II),(1,2-diaminocyclohexane)-cis(pyruvato)platinum(II), and(1,2-diaminocyclohexane)-oxalatoplatinum(II).
 11. The method of claim10, wherein said anti-tumor platinum-coordinator compound iscis-diamminedichloro-platinum(II).
 12. The method of claim 1, whereinsaid effective amount of D-methionine is in the range of from about 0.1mg/kg body weight to about 500 mg/kg body weight.
 13. The method ofclaim 1, wherein said effective amount of D-methionine is in the rangeof from about 1 mg/kg body weight to about 400 mg/kg body weight. 14.The method of claim 1, wherein said effective amount of D-methionine isin the range of from about 10 mg/kg body weight to about 300 mg/kg bodyweight.
 15. The method of claim 1, wherein said effective amount ofD-methionine is in the range of from about 1 mg/kg body weight to about100 mg/kg body weight.
 16. The method of claim 1, wherein said effectiveamount of D-methionine is in the range of from about 10 mg/kg bodyweight to about 75 mg/kg body weight.
 17. The method of claim 1, whereinsaid effective amount of D-methionine in relation to saidchemotherapeutic effective amount of said anti-tumorplatinum-coordination compound is in the range of from about 4:1 toabout 167:1, D-methionine:anti-tumor platinum-coordination compound, ona molar basis.
 18. The method of claim 1, wherein said effective amountof D-methionine in relation to said chemotherapeutic effective amount ofsaid anti-tumor platinum-coordination compound is in the range of fromabout 4.25:1 to about 100:1, D-methionine:anti-tumorplatinum-coordination compound, on a molar basis.
 19. The method ofclaim 1, wherein said effective amount of D-methionine in relation tosaid chemotherapeutic effective amount of said anti-tumorplatinum-coordination compound is in the range of from about 4.68:1 toabout 20:1, D-methionine:anti-tumor platinum-coordination compound, on amolar basis.
 20. The method of claim 1, wherein said effective amount ofD-methionine in relation to said chemotherapeutic effective amount ofsaid anti-tumor platinum-coordination compound is about 18.75:1,D-methionine:anti-tumor platinum coordination compound, on a molarbasis.
 21. The method of claim 1, wherein said D-methionine isadministered orally or parenterally.
 22. The method of claim 21, whereinsaid anti-tumor platinum-coordination compound administeredparenterally.
 23. The method of claim 22, wherein said parenteraladministration is by slow intravenous infusion.
 24. The method of claim1, further comprising administering to said patient a supplementalamount of D-methionine in the range of from about 0.1 mg/kg body weightto about 500 mg/kg body weight per week during and/or after the courseof treatment with said anti-tumor platinum-coordination.
 25. The methodof claim 24, wherein said supplemental amount of D-methionine isadministered orally or parenterally.
 26. The method of claim 1, whereinsaid anti-tumor platinum coordination compound is cisplatin.
 27. Themethod of claim 1, wherein an anti-ototoxic effective amount of amethionine protective agent consisting essentially of D-methionine isadministered to said patient.
 28. A method for preventing or reducingototoxicity in a patient undergoing treatment with a chemotherapeuticeffective amount of an anti-tumor platinum-coordination compoundselected from the group consisting of cisplatin, carboplatin, andiproplatin, comprising: intravenously administering to said patientabout 10 mg/kg body weight to about 75 mg/kg body weight ofD-methionine, or a pharmaceutically acceptable salt thereof, orD-methionine or a pharmaceutically acceptable salt thereof in a molarratio of about 18.75:1, D-methionine:anti-tumor platinum-coordinationcompound, within about one-half hour before administration of saidanti-tumor platinum-coordination compound to about one-half hour afteradministration of said anti-tumor platinum-coordination compound,wherein the D-methionine is substantially free of the L-isomer.
 29. Amethod for preventing or reducing weight loss in a patient selected fromthe group consisting of a human, a cat, and a dog undergoing treatmentwith a chemotherapeutic effective amount of an anti-tumorplatinum-coordination compound, comprising administering to said patientan anti-weight loss effective amount of D-methionine, wherein theD-methionine is substantially free of the L-isomer.
 30. The method ofclaim 29, wherein said anti-tumor platinum coordination compound iscisplatin.
 31. A method for preventing or reducing gastrointestinaltoxicity in a patient selected from the group consisting of a human, acat, and a dog undergoing treatment with a chemotherapeutic effectiveamount of an anti-tumor platinum-coordination compound, comprisingadministering to said patient an anti-gastrointestinal toxicityeffective amount of D-methionine, wherein the D-methionine issubstantially free of the L-isomer.
 32. The method of claim 31, whereinsaid anti-tumor platinum coordination compound is cisplatin.
 33. Amethod for preventing or reducing neurotoxicity in a patient selectedfrom the group consisting of a human, a cat, and a dog undergoingtreatment with a chemotherapeutic effective amount of an anti-tumorplatinum-coordination compound, comprising administering to said patientan anti-neurotoxicity effective amount of D-methionine, wherein theD-methionine is substantially free of the L-isomer.
 34. The method ofclaim 33, wherein said anti-tumor platinum coordination compound iscisplatin.
 35. A method for preventing or reducing alopecia in a patientselected from the group consisting of a human, a cat, and a dogundergoing treatment with a chemotherapeutic effective amount of ananti-tumor platinum-coordination compound, comprising administering tosaid patient an anti-alopecia effective amount of D-methionine, whereinthe D-methionine is substantially free of the L-isomer.
 36. The methodof claim 35, wherein said anti-tumor platinum-coordination compound iscisplatin.